This invention is directed to nuclease resistant oligonucleotides which are useful as therapeutics, diagnostics, and research reagents. Sugar-modified oligonucleotides which are resistant to nuclease degradation and are capable of modulating the activity of DNA and RNA are provided.
It has been recognized that oligonucleotides can be used to modulate mRNA expression by a mechanism that involves the complementary hybridization of relatively short oligonucleotides to mRNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific base pair hydrogen bonding of an oligonucleotide to a complementary RNA or DNA.
One deficiency of oligonucleotides for these purposes is their susceptibility to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly and extracellularly located. Unmodified, xe2x80x9cwild typexe2x80x9d, oligonucleotides are not useful as therapeutic agents because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been a focus of research directed towards the development of oligonucleotide therapeutics and diagnostics.
In addition to nuclease stability, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is a further important factor.
The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, is the temperature (in xc2x0 C.) at which 50% helical versus coil (unhybridized) forms are present. Tm is measured by using UV spectroscopy to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm the greater the strength of the binding of the nucleic acid strands.
Therefore, oligonucleotides modified to exhibit resistance to nucleases and to hybridize with appropriate strength and fidelity to its targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents and as oligonucleotide therapeutics. Various 2xe2x80x2-substitutions have been introduced in the sugar moiety of oligonucleotides. The nuclease resistance of these compounds has been increased by the introduction of 2xe2x80x2-substituents such as halo, alkoxy and allyloxy groups.
Ikehara et al. [European Journal of Biochemistry 139, 447 (1984)] have reported the synthesis of a mixed octamer containing one 2xe2x80x2-deoxy-2xe2x80x2-fluoroguanosine residue or one 2xe2x80x2-deoxy-2xe2x80x2-fluoroadenine residue. Guschlbauer and Jankowski [Nucleic Acids Res. 8, 1421 (1980)] have shown that the contribution of the 3xe2x80x2-endo increases with increasing electronegativity of the 2xe2x80x2-substituent. Thus, 2xe2x80x2-deoxy-2xe2x80x2-fluorouridine contains 85% of the C3xe2x80x2-endo conformer.
Furthermore, evidence has been presented which indicates that 2xe2x80x2-substituted-2xe2x80x2-deoxyadenosine polynucleotides resemble double-stranded RNA rather than DNA. Ikehara et al. [Nucleic Acids Res., 5, 3315 (1978)] have shown that a 2xe2x80x2-fluoro substituent in poly A, poly I, or poly C duplexed to its complement is significantly more stable than the ribonucleotide or deoxyribonucleotide poly duplex as determined by standard melting assays. Ikehara et al. [Nucleic Acids Res., 4, 4249 (1978)] have shown that a 2xe2x80x2-chloro or bromo substituent in poly(2xe2x80x2-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. [Biochemistry, 11, 4336 (1972)] have reported that poly(2xe2x80x2-chloro-2xe2x80x2-deoxyuridylic acid) and poly(2xe2x80x2-chloro-2xe2x80x2-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. [Nucleic Acids Research, 15, 6131 (1987)] have described the synthesis of mixed oligonucleotide sequences containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA heteroduplex (Tms, 49.0 and 50.1 versus 33.0 degrees for nonamers). Shibahara et al. [Nucleic Acids Research, 17, 239 (1987)] have reported the synthesis of mixed oligonucleotides containing 2xe2x80x2-OMe substituents on every nucleotide. The mixed 2xe2x80x2-OMe-substituted oligonucleotides were designed to inhibit HIV replication.
It is believed that the composite of the hydroxyl group""s steric effect, its hydrogen bonding capabilities, and its electronegativity versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2xe2x80x2-methoxy oligonucleotides is in the order of RNA-RNA greater than RNA-DNA greater than DNA-DNA.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligonucleotides comprising an RNA portion, bearing 2xe2x80x2-O-alkyl substituents, conjugated to a DNA portion via a phosphodiester linkage. However, being phosphodiesters, these oligonucleotides are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2xe2x80x2-O-substituted phosphorothioate oligonucleotides, including 2xe2x80x2-O-methylribooligonucleotide phosphorothioate derivatives. This application also discloses 2-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
European Patent application 260,032, filed Aug. 27, 1987, discloses oligonucleotides having 2xe2x80x2-O-methyl substituents on the sugar moiety. This application also makes mention of other 2xe2x80x2-O-alkyl substituents, such as ethyl, propyl and butyl groups.
International Publication Number WO 91/06556, published May 16, 1991, discloses oligomers derivatized at the 2-xe2x80x2 position with substituents, which are stable to nuclease activity. Specific 2xe2x80x2-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
European Patent application 399,330, filed May 15, 1990, discloses nucleotides having 2xe2x80x2-O-alkyl substituents. International Publication Number WO 91/15499, published Oct. 17, 1991, discloses oligonucleotides bearing 2xe2x80x2-O-alkyl, -alkenyl and -alkynyl substituents.
It has been recognized that nuclease resistance of oligonucleotides and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Oligonucleotides possessing nuclease resistance are also desired as research reagents and diagnostic agents.
In accordance with the present invention, compositions which are resistant to nuclease degradation and those that modulate the activity of DNA and RNA are provided. These compositions are comprised of sugar-modified oligonucleotides, which are specifically hybridizable with preselected nucleotide sequences of single-stranded or double-stranded target DNA or RNA. The sugar-modified oligonucleotides recognize and form double strands with single-stranded DNA and RNA.
The nuclease resistant oligonucleotides of the present invention consist of a single strand of nucleic acid bases linked together through linking groups. The oligonucleotides of this invention may range in length from about 5 to about 50 nucleic acid bases. However, in accordance with a preferred embodiment of this invention, a sequence of about 12 to 25 bases in length is optimal.
The individual nucleotides of the oligonucleotides of the present invention are connected via phosphorus linkages. Preferred phosphorous linkages include phosphodiester, phosphorothioate and phosphorodithioate linkages, with phosphodiester and phosphorothioate linkages being particularly preferred.
Preferred nucleobases of the invention include adenine, guanine, cytosine, uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
In accordance with this invention at least one of the 2xe2x80x2-deoxyribofuranosyl moiety of at least one of the. nucleosides of an oligonucleotide is modified. A halo, alkoxy, aminoalkoxy, alkyl, azido, or amino group may be added. For example, F, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, SMe, SO2Me, ONO2, NO2, NH3, NH2, NH-alkyl, OCH2CHxe2x95x90CH2 (allyloxy), OCH3xe2x95x90CH2, OCCH, where alkyl is a straight or branched chain of C1 to C20, with unsaturation within the carbon chain.
The present invention also includes oligonucleotides formed from a plurality of linked-xcex2-nucleosides including 2xe2x80x2-deoxy-erythro-pentofuranosyl-xcex2-nucleosides. These nucleosides are connected by charged phosphorus linkages in a sequence that is specifically hybridizable with a complementary target nucleic acid. The sequence of linked nucleosides is divided into at least two subsequences. The first subsequence includes xcex2-nucleosides, having 2xe2x80x2-substituents, linked by charged 3xe2x80x2-5xe2x80x2 phosphorous linkages. The second subsequence consists of 2xe2x80x2-deoxy-erythro-pentofuranosyl-xcex2-nucleosides linked by charged 3xe2x80x2-5xe2x80x2 phosphorous linkages bearing a negative charge at physiological pH. In further preferred embodiments there exists a third subsequence whose nucleosides are selected from those selectable for the first subsequence. In preferred embodiments the second subsequence is positioned between the first and third subsequences. Such oligonucleotides of the present invention are also referred to as xe2x80x9cchimericxe2x80x9d or xe2x80x9cgappedxe2x80x9d oligonucleotides, or xe2x80x9cchimeras.xe2x80x9d
The resulting novel oligonucleotides of the invention are resistant to nuclease degradation and exhibit hybridization properties of higher quality relative to wild-type DNA-DNA and RNA-DNA duplexes and phosphorus-modified oligonucleotide duplexes containing methylphosphonates, phophoramidates and phosphate triesters.
The invention is also directed to methods for modulating the production of a protein by an organism comprising contacting the organism with a composition formulated in accordance with the foregoing considerations. It is preferred that the RNA or DNA portion which is to be modulated be preselected to comprise that portion of DNA or RNA which codes for the protein whose formation is to be modulated. Therefore, the oligonucleotide to be employed is designed to be specifically hybridizable to the preselected portion of target DNA or RNA.
This invention is also directed to methods of treating an organism having a disease characterized by the undesired production of a protein. This method comprises contacting the organism with a composition in accordance with the foregoing considerations. The composition is preferably one which is designed to specifically bind with mRNA which codes for the protein whose production is to be inhibited.
The invention further is directed to diagnostic methods for detecting the presence or absence of abnormal RNA molecules, or abnormal or inappropriate expression of normal RNA molecules in organisms or cells.
The invention is also directed to methods for the selective binding of RNA for use as research reagents and diagnostic agents. Such selective and strong binding is accomplished by interacting such RNA or DNA with oligonucleotides of the invention which are resistant to degradative nucleases and which display greater fidelity of hybridization than any other known oligonucleotide.